The present invention resides in the technical fields of immunology and enzymology.
The cytochrome P450 family of enzymes is primarily responsible for the metabolism of xenobiotics such as drugs, carcinogens and environmental chemicals, as well as several classes of endobiotics such as steroids and prostaglandins. Members of the cytochrome P450 family are present in varying levels and their expression and activities are controlled by variables such as chemical environment, sex, developmental stage, nutrition and age.
More than 200 cytochrome P450 genes have been identified. There are multiple forms of these P450 and each of the individual forms exhibit degrees of specificity towards individual chemicals in the above classes of compounds. In some cases, a substrate, whether it be drug or carcinogen, is metabolized by more then one of the cytochromes P450.
Human cytochrome P450 1A2 constitutes about 13% of total P450 in human liver and is the second most abundant P450 following human cytochrome P450 3A4 (Vermeulen, 1996). P450 1A2 catalyzes the metabolism of a large variety of drugs and carcinogens (Rendic et al., 1997). Drugs metabolized by human P450 1A2 include phenacetin, R-warfarin, clomipramine, imipramine, theophyline, theobromine, paraxanthine, caffeine, chlorzoxazone, 7-methoxyresorufin, and 7-ethoxycoumarin. P450 1A2 is also has a major role in activating mutagens and carcinogens. For example, 1A2 metabolically activates the food pyrolysis products IQ and MeIQx to active mutagens (Edwards et al., 1994; Rendic et al., 1997).
Genetic polymorphisms of cytochromes P450 result in phenotypically-distinct subpopulations that differ in their ability to perform biotransformations of particular drugs and other chemical compounds. These phenotypic distinctions have important implications for selection of drugs. For example, a drug that is safe when administered to most humans may cause toxic side-effects in an individual suffering from a defect in an enzyme required for detoxification of the drug. Alternatively, a drug that is effective in most humans may be ineffective in a particular subpopulation because of lack of a enzyme required for conversion of the drug to a metabolically active form. Further, individuals lacking a biotransformation enzyme are often susceptible to cancers from environmental chemicals due to inability to detoxify the chemicals. Eichelbaum et al., Toxicology Letters 64/65, 155-122 (1992). Accordingly, it is important to identify individuals who are deficient in a particular P450 enzyme, so that drugs known or suspected of being metabolized by the enzyme are not used, or used only with special precautions (e.g., reduced dosage, close monitoring) in such individuals. Identification of such individuals may indicate that such individuals be monitored for the onset of cancers.
Existing methods of identifying deficiencies in patients are not entirely satisfactory. Patient metabolic profiles are often assessed with a bioassay after a probe drug administration. Individuals with below normal cytochrome P450 activity exhibit physiologic accumulation of unmodified drug and have a high metabolic ratio of probe drug to metabolite. This bioassay has a number of limitations: lack of patient cooperation, adverse reactions to probe drugs, and inaccuracy due to coadministration of other pharmacological agents or disease effects. See, e.g., Gonzalez et al., Clin. Pharmacokin. 26, 59-70 (1994). Genetic assays by RFLP (restriction fragment length polymorphism), ASO PCR (allele specific oligonucleotide hybridization to PCR products or PCR using mutant/wild-type specific oligo primers), SSCP (single stranded conformation polymorphism) and TGGE/DGGE (temperature or denaturing gradient gel electrophoresis), MDE (mutation detection electrophoresis) are time-consuming, technically demanding and limited in the number of gene mutation sites that can be tested at one time.
A complication in patient drug choice is that most drugs have not been characterized for their metabolism by P450 1A2 and other cytochromes P450. Without knowing which cytochrome(s) p450 is/are responsible for metabolizing an individual drug, an assessment cannot be made for the adequacy of a patient""s P450 profile. For such drugs, there is a risk of adverse effects if the drugs are administered to deficient metabolizers.
Monoclonal antibodies that specifically bind to 1A2 and inhibit its activity, if available, could be used to screen drugs for their metabolism by 1A2 and/or identify 1A2 deficient metabolizers by simple bioassays, thereby overcoming the problems in prior complicated methods discussed above. However, such monoclonal antibodies represent, at best, a small subset of the total repertoire of antibodies to human cytochrome P450 1A2, and have not hitherto been isolated. Although in polyclonal sera, many classes of antibody may contribute to inhibition of enzyme activity of P450 1A2 as a result of multiple antibodies in sera binding to the same molecule of enzyme, only a small percentage of these, if any, can inhibit as a monoclonal. A monoclonal antibody can inhibit only by binding in such a manner that it alone block or otherwise perturb the active site of an enzyme. The existence and representation of monoclonal antibodies with inhibitory properties thus depend on many unpredictable factors. Among them are the size of the active site in an enzyme, whether the active site is immunogenic, and whether there are any sites distal to the active site that can exert inhibition due to stearic effects of antibody binding. The only means of obtaining antibodies with inhibitory properties is to screen large numbers of hybridoma until one either isolates the desired antibody or abandons the task through failure.
Notwithstanding these difficulties, the present invention provides inter alia monoclonal antibodies that specifically bind to human cytochrome P450 1A2 and inhibit its activity.
The invention provides isolated binding agents that compete with a monoclonal antibody selected from the group consisting of MAb 26-7-5, MAb 951-5-1, and MAb 1812-4-8 for specific binding to human cytochrome p450 1A2, and that specifically inhibit 1A2-catalyzed metabolism of phenacetin by at least 50%. Preferred binding agents are monoclonal antibodies. Some binding agents lacks specific binding to at least one cytochrome P450 selected from the group consisting of human cytochromes P450 1A1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5. Some binding agents lack specific binding to each of human cytochromes P450 1A1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5. Preferred binding agents are able specifically to inhibit the enzyme activity of human cytochrome p450 1A2 by at least 80%. Some binding agents are binding fragments, such as Fab fragments.
MAb 26-7-5, MAb 951-5-1, and MAb 1812-4-8 are exemplified monoclonal antibodies. Some other monoclonal antibodies are analogs of these monoclonal antibodies comprising a light chain variable domain having at least 80% sequence identity with the light chain variable domain of a monoclonal antibody selected from the group consisting of MAb 26-7-5, MAb 951-5-1, and MAb 1812-4-8, wherein the percentage sequence identity is determined by aligning amino acids in the light chain variable domains by the Kabat numbering convention and a heavy chain variable domain having at least 80% sequence identity with the heavy chain variable domain of a monoclonal antibody selected from the group, wherein the percentage sequence identity is determined by aligning amino acids in the heavy chain variable domains by the Kabat numbering convention.
The invention further provides cell lines producing monoclonal antibodies as described above. Cells lines can be eucaryotic or procaryotic.
The invention further provides methods of determining whether cytocbrome P450 1A2 metabolizes a compound. Such methods entail contacting the compound with cytochrome P450 1A2 in the presence of varying amounts of the binding agent of claim 1. Metabolism of the compound is then assayed as a function of amount of binding agent, a decrease of metabolism with amount of binding agent indicating that cytochrome P450 1A2 metabolizes the compound. In some such methods, the compound is contacted with cytochrome P450 1A2 in a sample containing a collection of cytochrome P450 enzymes including 1A2.
In some methods, the sample is a tissue sample. In some methods, the collection of enzymes are obtained from a cell culture expressing the enzymes. In some methods, the compound is a drug, steroid or carcinogen.
The invention further provides methods of detecting cytochrome p450 1A2. Such methods entail contacting a sample suspected of containing cytochrome P450 1A2 with a binding agent described above. One then determines whether the agent specifically binds to the sample, specific binding indicating the presence of cytochrome P450 1A2 in the sample.
The invention further provides methods of measuring p450 1A2 levels in an individual relative to p450 1A2 levels in a control population. Such methods entail contacting a sample suspected of containing cytochrome P450 1A2 from the individual and a substrate of 1A2. One then determines the p450 1A2 levels in the individual relative to p450 1A2 levels in the control population.